A major goal of HIV vaccine development is to create vaccines that elicit broadly neutralizing antibodies (NAbs) to the gp120/gp41 envelope (Env) spikes on virions. Efforts to accomplish this goal have met with three obstacles: (1) it is difficult to replicate the structure of gp120/gp41 trimers in a vaccine; (2) NAbs should be at the highest possible titer on exposed mucosal surfaces; and (3) extensive variation in Env has made it difficult to find the best Env sequence for use in a vaccine. This project will study innovative ways to overcome these roadblocks through three Aims. Aim 1 will use intramuscular DNA vaccination with an Env plasmid along with a plasmid to generate CD8+ T cells that are cytotoxic for the transfected muscle cells. This strategy utilizes the ability of plasmid DNA to express Env in its correctly folded native conformation on the membranes of cells at the vaccination site. The addition of a DNA vaccine for cytotoxic CD8+ T cells will enable cell debris expressing native Env to reach B cells in the draining lymph nodes, based on the previously unexploited finding that CD8+ T cells markedly enhance antibody responses to DNA vaccines for nonsecreted antigens. We call this approach 'CD8+ T cell-mediated Antibody-Eliciting Vaccine' (CAEVac). Aim 2 will test the abilty of DNA vaccines encoding high epitope density forms of Env to augment the anti-Env antibody response. Aim 3 will test the adjuvant effects of CD40L, GITRL, BAFF, and APRIL, four TNF superfamily ligands, on the antibody responses to the DNA vaccines of Aim 1. BAFF in particular has potential as an adjuvant for mucosal IgA responses. When this innovation project is complete, new concepts for vaccines that elicit HIV NAbs will have been evaluated for advancement to further studies in the HIV vaccine pipeline. This project will develop new immunostimulants that could significantly improve the strength of viral vaccines. While this research is focused on HIV, the same immunostimulants could be applied to vaccines against many other infections including influenza, viral hepatitis, West Nile virus, smallpox, and agents of biodefense significance. [unreadable] [unreadable] [unreadable]